Synthesis of new coumarin 3-(N-aryl) sulfonamides and their anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2004.05.016

Synthesis of coumarin 3-(N-aryl) sulfonamides was accomplished either by Knoevenagel condensation of anilinosulfonylacetic acids with suitable salicylaldehydes or by the reaction of methyl anilinosulfonylacetates with substituted salicylaldehydes in presence of a catalytic amount of a base. All the compounds tested for antiproliferative activity in different cancer cell lines have shown GI₅₀ values less than 100μM.

Full text of DOI:10.1016/j.bmcl.2004.05.016

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4093–4097
Synthesis of new coumarin 3-(N-aryl) sulfonamides and
their anticancer activity
Natala Srinivasa Reddy, Muralidhar Reddy Mallireddigari, Stephen Cosenza,
Kiranmai Gumireddy, Stanley C. Bell, E. Premkumar Reddy and M. V. Ramana Reddy*
Fels Institute for Cancer Research, Temple University School of Medicine, 3307 North Broad Street,
Philadelphia, PA 19140-5101, USA
Received 26 March 2004; accepted 10 May 2004
Available online 17 June 2004
Abstract—Synthesis of coumarin 3-(N-aryl) sulfonamides was accomplished either by Knoevenagel condensation of anilinosulfon-
ylacetic acids with suitable salicylaldehydes or by the reaction of methyl anilinosulfonylacetates with substituted salicylaldehydes in
presence of a catalytic amount of a base. All the compounds tested for antiproliferative activity in different cancer cell lines have
shown GI50 values less than 100 lM.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The diverse biological activities of natural and synthetic
coumarin derivatives as anticoagulants and antithrom-
botics are well known.1 Some of the coumarin deriva-
tives are also reported as triplet sensitizers,2 anti-HIV3
agents, lipid-lowering4 agents, and antioxidants.5 They
have also been found to inhibit lipid peroxidation and to
possess vasorelaxant,6 anti-inflammatory,7 and anti-
cancer activity.8 Many coumarin derivatives are also
known as free radical scavengers9 and two naturally
occurring coumarins have been found to exhibit cyto-
toxicity against a panel of mammalian cancer cell lines.10
Some coumarin-3-carboxamides were reported as potent
inhibitors of proteases such as a-chymotrypsin (CT)11–13
and human leukocyte elastase (HLE).14–16 Recently, a
new class of sulfonamides17;18 has been used in the
treatment of diseases arising from abnormal cell growth
and proliferation. Most cancers are characterized by
uncontrolled cell proliferation, lack of cell differentia-
tion, and loss of contact inhibition, which confers upon
the tumor cell a capability to invade local tissues and
metastasize. Recently, a series of arylsulfonanilides has
been reported as potent inhibitors of various cancer cells
at very low concentrations.19 Based on the diverse bio-
logical activities of the coumarins and aryl sulfon-
amides, we have designed and synthesized a series of
novel compounds that have both coumarin and sulfon-
amide entities in one molecule and have evaluated them
for their antitumor activity.
Although several 3-substituted coumarins are known in
literature,20–23 very little information is available on
coumarin-3-sulfonamides. In earlier papers,20–23 differ-
ent authors have used the same procedure for the syn-
thesis of coumarin-3-sulfonamides where they have
condensed coumarin-3-sulfonyl chloride with different
aromatic amines. This method of synthesis has a limi-
tation in having a variety of substituents at different
positions on both aromatic rings and may not be useful
in creating a library of new broadly unsubstituted cou-
marin-3-arylsulfonamides. In this letter we report a
novel route for the synthesis of coumarin-3-sulfon-
amides using the Knoevenagel type of condensation
reaction.
The reaction of methyl-2-chlorosulfonyl acetate with
aromatic amines (1) gave methyl anilinosulfonylace-
tates (2), which on hydrolysis yielded anilinosulfonyl-
acetic acids (3). Condensation of anilinosulfonylacetic
acids with substituted salicylaldehydes (4) in glacial
acetic acid in the presence of a catalytic amount of
benzylamine gave coumarin-3-sulfonamides (5) in
quantitative yields24a (Scheme 1). Alternatively, couma-
rin-3-sulfonamides (5) were also prepared by reacting
Keywords: Coumarins; Sulfonamides; Jun kinase.
* Corresponding author. Tel.: +1-215-707-7336; fax: +1-215-707-1454;
e-mail: rreddy@temple.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.016

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N. Srinivasa Reddy et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4093–4097
O
leading to the formation of coumarin-3-sulfonamides. A
noncyclized Knoevenagel reaction would give an a,b-
unsaturated compound with vinylic protons splitting as
doublets at d 7.3 and 7.6. Therefore, a singlet for a
methine proton at d 8.50–9.20 confirmed the cyclization
reaction with the formation of coumarin-3-sulfon-
amides.
O
O
S
O
S
2
2
NH
2
HN
OCH
b
HN
OH
3
a
X
X
X
1
2
3
The effect of coumarin-3-sulfonamides on the growth of
human tumor cells in culture was evaluated using
androgen receptor negative prostate (DU145), colo-
rectal (DLD-1), nonsmall cell lung carcinoma (H157),
Estrogen receptor negative breast (BT20), and chronic
myeloid leukemia (K562) cell lines. The dose response of
each cell line was established by determining the number
of viable cells after 96 h of continuous treatment against
five different concentrations (1–100 lM range) of each
compound. Table 1 shows the GI50 values, the concen-
tration of each compound required to inhibit the growth
of each tumor cell line by 50%, determined for each
compound in the series.
X
O2
CHO
OH
S
c
N
H
+
3
O
O
Y
Y
4
5
Scheme 1. Reagents and conditions: (a) ClSO2CH2COOCH3, triethyl-
amine/THF, N2, rt, 3 h; (b) 10% NaOH in H2O; (c) CH3COOH,
C6H5CH2NH2.
X
O2
S
CHO
OH
N
Extra cellular signals received at transmembrane recep-
tors are relayed into cells by the signal transduction
pathways that have been implicated in induction of cell
proliferation,26 differentiation,27 and apoptosis.28 The
mitogen activated protein kinase (MAPK) cascade is a
major signaling system and many steps in this pathway
are well conserved in different species. The best-studied
MAPK family members are extra cellular signal-regu-
lated kinases (ERKs)29 and c-Jun NH2 terminal kinases
(JNKs).30 JNKs are the members of the class of stress
activated protein kinases (SAPK) and are shown to be
activated by treatment of cells with UVradiation, pro-
inflammatory cytokines and environmental stress.31 It
has been shown that activation of ERK involves phos-
phorylation of threonine and tyrosine residues by the
upstream dual kinase and this activation signals cell
proliferation where as the activation of JNKs eventually
leads to cell growth inhibition and apoptosis.32
a
H
2
+
O
O
Y
Y
5
4
Scheme 2. Reagents and conditions: (a) piperidine, ethanol, reflux,
5 min.
methyl anilino-sulfonylacetates with substituted salicyl-
aldehydes in the presence of piperidine and ethanol24b
(Scheme 2).
All the new compounds synthesized were characterized
by 1H NMR spectroscopy. Elemental analysis values for
C, H, and N were within the range of the theoretical
value. The H NMR spectra of all new products (5a–j)
showed a characteristic peak as a singlet at d 8.50–9.20
for a methine proton ascertaining a cyclization reaction
1
Table 1. Growth inhibition of cultured tumor cells
X
O2
S
N
H
O
O
Y
5
Compd
Cell line (GI50 lM)a
X
Y
BT20
DU145
H157
DLD-1
K562
5a
5b
5c
5d
5e
5f
4-OMe
4-OMe
4-OMe
4-OMe, 3-OH
4-F, 3-NH2
4-F, 3-NH2
4-Br
8-Br
6-Cl
16
27
52
65
54
68
18
64
17
12
15
22
57
42
48
56
25
52
14
12
18
28
74
76
72
78
32
74
16
15
20
32
78
82
80
92
38
85
18
14
14
23
48
51
42
44
27
58
11
16
8-OEt
6-Cl
8-OEt
6-OMe
6-OMe
8-OEt
8-Cl
5g
5h
5i
4-Br
4-Br
5j
4-Br
8-Br
a The GI50 was determined25 by direct extrapolation from each dose response curve.

N. Srinivasa Reddy et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4093–4097
4095
Since many types of cancer cells, following treatment
with coumarin-3-sulfonamides, have shown consider-
able growth inhibition followed by cell death (Table 1),
we evaluated the role of these compounds in activating
Jun kinase signaling pathway. Some of the compounds,
which have higher potency (5a, 5b, 5g, 5h, and 5i) were
added to BT-20 cells at 50 and 100 lM concentration
and harvested after 12 h. The cell lysates were then
analyzed for c-Jun kinase activity33 by immunoprecipi-
tating JNK1 and assessing the ability of these immu-
noprecipitates to phosphorylate GST-linked c-Jun.
Results33 from these studies demonstrate that GST-c-
Jun is hyperphosphorylated in all the cells that were
treated with coumarin-3-sulfonamides compared to the
DMSO treated controls (Fig. 1).
analysis: calcd C 46.84%; H 2.95%; N 3.41%. Found C
46.67%; H 2.74%; N 3.36%.
6-Chlorocoumarin 3-(N-4-methoxyphenyl)sulfonamide
(5b): Mp 175–177 ꢁC; 1H NMR (500 MHz, DMSO):
10.24 (br s, 1H), 8.72 (s, 1H), 8.06 (s, 1H), 7.77 (d,
J ¼ 9:0 Hz, 1H), 7.49 (d, J ¼ 9:5 Hz, 1H), 7.04 (d,
J ¼ 7:0 Hz, 2H), and 6.79 (d, J ¼ 7:0 Hz, 2H); C, H, N
analysis: calcd C 52.54%; H 3.30%; N 3.83%. Found C
52.33%; H 3.26%; N 3.77%.
8-Ethoxycoumarin 3-(N-4-methoxyphenyl)sulfonamide
1
(5c): Mp 196–198 ꢁC; H NMR (500 MHz, DMSO):
10.18 (br s, 1H), 8.71 (s, 1H), 7.45 (d, J ¼ 8:0 Hz, 1H),
7.40 (d, J ¼ 8:0 Hz, 1H), 7.29 (d, J ¼ 8:0 Hz, 1H), 7.05
(d, J ¼ 7:0 Hz, 2H), 6.79 (d, J ¼ 7:0 Hz, 2H), 4.13 (q,
J ¼ 7:0 Hz, 2H), 3.61 (s, 3H), and 1.36 (t, J ¼ 6:9 Hz,
3H); C, H, N analysis: calcd C 57.59%; H 4.56%; N
3.73%. Found C 57.33%; H 4.38%; N 3.65%.
Several authors have reported the role of activated
JNKs in inhibition of cell growth and death.28;34;35 This
pathway involves extra cellular signal-regulated kinases,
which constitutes the PAK/MEKK/SEK/JNK kinase
cascade, which ultimately phosphorylates c-Jun that is
involved in apoptotic pathway. The activation of JNK1
by coumarin-3-sulfonamides as shown in immune
complex kinase assay (Fig. 1), clearly shows that these
compounds activate JNK pathway either by interacting
with JNK1 or with one of the upstream kinases in this
pathway. Current efforts are focused on identifying the
target kinase for these compounds.
6-Chlorocoumarin 3-(N-4-methoxy 3-hydroxyphenyl)-
1
sulfonamide (5d): Mp 225–226 ꢁC; H NMR (400 MHz,
DMSO): 10.16 (br s, 1H), 9.14 (s, 1H), 8.76 (s, 1H), 8.12
(s, 1H), 7.81 (dd, J ¼ 8:8, 3.2 Hz, 1H), 7.53 (d,
J ¼ 8:8 Hz, 1H), 6.77 (d, J ¼ 8:8 Hz, 1H), 6.65 (d,
J ¼ 2:5 Hz, 1H), 6.55 (dd, J ¼ 8:8, 2.5 Hz, 2H), and 3.67
(s, 3H); C, H, N analysis: calcd C 50.33%; H 3.17%; N
3.67%. Found C 50.18%; H 2.95%; N 3.61%.
In summary, we report here a series of new coumarin-3-
sulfonamides prepared by a novel method and their
ability to activate JNK1 and kill tumor cells in vitro.
These results provide an approach to develop new
potent coumarin sulfonamides that kill tumor cells by
activating JNK pathway.
8-Ethoxycoumarin 3-(N-4-fluoro 3-aminophenyl)sulfon-
amide (5e): Mp 160–162 ꢁC; 1H NMR (400 MHz,
DMSO): 10.80 (br s, 1H), 8.71 (s, 1H), 7.54 (d,
J ¼ 7:7 Hz, 1H), 7.50 (d, J ¼ 7:7 Hz, 1H), 7.42 (d,
J ¼ 7:7 Hz, 1H), 6.84 (d, J ¼ 8:8 Hz, 1H), 6.60 (d,
J ¼ 8:8 Hz, 1H), 6.31 (m, 1H), 5.10 (br s, 2H), 4.28 (q,
J ¼ 7:0 Hz, 2H), and 1.51 (t, J ¼ 6:9 Hz, 3H).
8-Bromocoumarin 3-(N-4-methoxyphenyl)sulfonamide
(5a): Mp 182–184 ꢁC; 1H NMR (500 MHz, DMSO):
10.24 (br s, 1H), 8.71 (s, 1H), 8.18 (s, 1H), 7.87 (d,
J ¼ 9:0 Hz, 1H), 7.41 (d, J ¼ 9:0 Hz, 1H), 7.04 (d,
J ¼ 9:0 Hz, 2H), and 6.79 (d, J ¼ 7:0 Hz, 2H); C, H, N
6-Methoxycoumarin 3-(N-4-fluoro 3-aminophenyl)sul-
fonamide (5f): Mp 255–260 ꢁC; 1H NMR (500 MHz,
DMSO): 10.18 (br s, 1H), 8.53 (s, 1H), 7.52 (d,
J ¼ 2:8 Hz, 1H), 7.43 (d, J ¼ 9:0 Hz, 1H), 7.33 (dd,
J ¼ 9:0, 2.8 Hz, 1H), 6.70 (d, J ¼ 8:5 Hz, 1H), 6.47 (d,
J ¼ 8:5 Hz, 1H), 6.20 (m, 1H), 4.79 (br s, 2H), and 3.89
(s, 3H).
6-Methoxycoumarin 3-(N-4-bromophenyl)sulfonamide
(5g): Mp 166–168 ꢁC; 1H NMR (400 MHz, DMSO):
10.88 (br s, 1H), 9.02 (s, 1H), 7.73 (d, J ¼ 2:5 Hz, 1H),
7.60–7.50 (m, 4H), 7.27 (d, J ¼ 7:0 Hz, 2H), and 3.95 (s,
3H); C, H, N analysis: calcd C 46.84%; H 2.95%; N
3.41%. Found C 46.70%; H 2.94%; N 3.12%.
8-Ethoxycoumarin
3-(N-4-bromophenyl)sulfonamide
(5h): Mp 206–209 ꢁC; 1H NMR (400 MHz, DMSO):
10.75 (br s, 1H), 8.91 (s, 1H), 7.52 (d, J ¼ 7:6 Hz, 1H),
7.45 (d, J ¼ 8:8 Hz, 3H), 7.36 (t, J ¼ 8:0 Hz, 1H), 7.13
(d, J ¼ 8:0 Hz, 2H), 4.17 (q, J ¼ 7:0 Hz, 2H), and 1.39
(t, J ¼ 6:9 Hz, 3H); C, H, N analysis: calcd C 48.13%; H
3.32%; N 3.30%. Found C 47.60%; H 3.19%; N 3.21%.
Figure 1. Activation of JNK-1 by coumarin-3-sulfonamides. BT-20
cell were treated with 50 and 100 lM of coumarin-3-sulfonamides and
incubated for 12 h before cell lysis and immunoprecipitation with
JNK1 antibody. The kinase activity of the immunoprecipitated JNK1
was tested using GST fused c-Jun (1-79 AA) as a substrate.
8-Chlorocoumarin 3-(N-4-bromophenyl)sulfonamide (5i):
Mp 202–204 ꢁC; 1H NMR (400 MHz, DMSO): 10.80 (br
s, 1H), 8.89 (s, 1H), 8.13 (s, 1H), 7.81 (dd, J ¼ 8:8,

4096
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2.8 Hz, 1H), 7.51 (d, J ¼ 8:8 Hz, 1H), 7.44 (d,
J ¼ 8:8 Hz, 2H), 7.11 (d, J ¼ 8:8 Hz, 2H), 6.79 (d,
J ¼ 7:0 Hz, 2H), 4.13 (q, J ¼ 7:0 Hz, 2H), and 1.36 (t,
J ¼ 6:9 Hz, 3H); C, H, N analysis: calcd C 43.45%; H
2.19%; N 3.38%. Found C 43.39%; H 2.09%; N 3.21%.
19. Rubenstein, S. M.; Baichwal, V.; Beckmann, H.; Clark, D.
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8-Bromocoumarin 3-(N-4-bromophenyl)sulfonamide (5j):
Mp 206–208 ꢁC; 1H NMR (400 MHz, DMSO): 10.80 (br
s, 1H), 8.90 (s, 1H), 8.26 (s, 1H), 7.93 (d, J ¼ 9:0 Hz,
1H), 7.45 (d, J ¼ 8:8 Hz, 3H), 7.11 (d, J ¼ 8:8 Hz, 2H),
7.05 (d, J ¼ 7:0 Hz, 2H), 6.79 (d, J ¼ 7:0 Hz, 2H), 4.13
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24. (a) General experimental procedure for the synthesis of
title compounds: Into a R.B. flask were added ani-
linosulfonyl acetic acid (1 equiv), salicylaldehyde (1 equiv)
dissolved in acetic acid (10 mL), and a catalytic amount of
benzyl amine (0.001 equiv), and the contents were refluxed
for about 8–12 h in an oil bath. After cooling, the
precipitated product was filtered and washed with isopro-
panol and diethyl ether. The pure products obtained were
submitted for biological assay; (b) General method: To a
solution of substituted salicylaldehydes (10 mmol) in warm
absolute ethanol (20 mL) was added methyl anilinosulfo-
nyl acetate (11 mmol) and three drops of piperidine. The
solution was heated under reflux for 5 min. The crystalline
product obtained after cooling was separated by filtration
and washed three times with absolute ethanol to obtain a
pure coumarin sulfonamide.
Acknowledgements
The authors wish to thank Fels Foundation and
Onconova Therapeutics Inc., New Jersey for their
financial support and encouragement.
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N. Srinivasa Reddy et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4093–4097
4097
twice with lysis buffer and twice with JNK buffer (20 mM
HEPES, pH 7.6, 20 mM b-glycerophosphate, 10 mM
MgCl2, and 100 lM Na3VO4. The kinase reaction was
carried out by resuspending the beads in 40 lL of JNK
buffer containing 20 lM [c-32P]ATP (5000 CPM/pmol) and
incubating them for 20 min at 30 ꢁC using 5 lg GST-c-Jun
as substrate. After stopping the reaction with the addition
of Laemmli’s buffer followed by boiling the samples for
3 min, the phosphorylated GST-c-Jun was separated on
12% SDS-PAGE. The gel was dried, and an autoradio-
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